Improved diagnosis of Chlamydia trachomatis (Ct) infections represents a critical unmet medical need. Ct is the most common cause of bacterial sexually transmitted diseases (STD) worldwide, and the Centers for Disease Control and Prevention estimate that there are 2.8 million US cases annually. Currently, 40% of women with untreated infection will develop pelvic inflammatory disease (PID), 20% of whom will become infertile, 18% will experience debilitating, chronic pelvic pain, and 9% will have a life-threatenig ectopic pregnancy. If an infected pregnant woman is not treated, her baby has a 50% chance of developing conjunctivitis and a 20% chance of pneumonia in the first six months of life. Ct is also a risk factor for invasive squamous-cell carcinoma of the cervix and a complicating factor in HIV-1 infection and transmission. In males, the clinical presentation includes proctitis, genital ulcer and/or inguinal lymphadenopathy. The main obstacle to stemming this epidemic is the lack of an inexpensive nucleic acid-based point-of-care (POC) diagnostic for screening. Current chlamydial diagnostics are primarily based on nucleic acid amplification tests (NAAT) that lack concordance across the different NAATs, vary in sensitivity (although specificity is high), are expensive, require technical expertise, take days for results, and cannot be performed at the POC. They are also unable to differentiate between invasive lymphogranuloma venereum (LGV) versus non-invasive strains, the former of which require weeks of antibiotic therapy for eradication. Our overall SBIR goal is to develop a rapid, cost-effective, sensitive and specific Ct POC diagnostic system to increase early detection, inform appropriate treatment, reduce the rate of infections, and thereby reduce sequelae. In SBIR Phase I, we developed an initial microfluidic NAAT and demonstrated that its sensitivity and specificity are superior to those of a commercial NAAT. In SBIR Phase II, we propose to: 1) Optimize our assay based on information gained by whole genome sequencing clinical Ct strains; 2) incorporate all assay processes in an easy-to operate breadboard instrument; and 3) do a head-to-head comparison of the optimized system against two commercially available assays. Given the genomic expertise of Dr. Tim Read and the chlamydial STD expertise of Dr. Dean, and the molecular biological and microfluidic expertise of Dr. Selden, the application provides a unique collaborative opportunity to finally obtain a rapid nucleic-acid based POC diagnostic for C. trachomatis.